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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

The NFκBp50 RHD binds the MYB SLR polyU RNA via a lysine-rich region.(A) Sequence alignment of the RHD of NFκB proteins. Black boxes indicate amino acid identity and grey boxes indicate similarity. The numbers refer to the first position of the segments within the respective proteins. The asterisk refers to residues that were analyzed by mutagenesis. (B) Wild type (wt) and mutant NFκBp50 RHDs analyzed by Coomassie staining. (C) Radiolabeled RNA probe generated from pBluescript II KS MYB SLR polyU template was incubated with 12.5 ng of recombinant wt or mutant NFκBp50 RHD as indicated and the reactions resolved on a 5% Tris-glycine gel. The black arrow indicates the position of the NFκBp50 RHD-MYB SLR polyU RNA complex; the grey arrow indicates free probe.
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pone.0122919.g003: The NFκBp50 RHD binds the MYB SLR polyU RNA via a lysine-rich region.(A) Sequence alignment of the RHD of NFκB proteins. Black boxes indicate amino acid identity and grey boxes indicate similarity. The numbers refer to the first position of the segments within the respective proteins. The asterisk refers to residues that were analyzed by mutagenesis. (B) Wild type (wt) and mutant NFκBp50 RHDs analyzed by Coomassie staining. (C) Radiolabeled RNA probe generated from pBluescript II KS MYB SLR polyU template was incubated with 12.5 ng of recombinant wt or mutant NFκBp50 RHD as indicated and the reactions resolved on a 5% Tris-glycine gel. The black arrow indicates the position of the NFκBp50 RHD-MYB SLR polyU RNA complex; the grey arrow indicates free probe.

Mentions: Within the RHD we pinpointed two motifs (aa 139–141; LGI) and (aa 146–148; KKK) that were highly conserved amongst the various human and mouse NFκB family members (Fig 3A). Small clusters of highly basic amino acids rich in Lys residues are features of proteins that bind to RNA, with the positive charge of these residues complementing the negative charged surface of RNA [34]. We therefore sought to determine if the KKK residues contributed to the MYB SLR polyU RNA binding activity of NFκBp50. NFκBp50 RHD mutants were generated in which the Lys residues were substituted singly or collectively with Ala or Asp (Fig 3B). Single substitution of the Lys residues 146, 147 or 148 subtly reduced NFκBp50 RHD binding to MYB SLR polyU RNA (Fig 3C). In contrast, binding of p50 RHD was substantially reduced by substituting all three Lys for Ala or Asp (Fig 3C). The LGI residues were also each substituted for Ala (Fig 3B). Substituting the Leu or Ile residues reduced NFκBp50 binding to MYB SLR polyU RNA probe while substituting the Gly had little effect (Fig 3C).


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)

The NFκBp50 RHD binds the MYB SLR polyU RNA via a lysine-rich region.(A) Sequence alignment of the RHD of NFκB proteins. Black boxes indicate amino acid identity and grey boxes indicate similarity. The numbers refer to the first position of the segments within the respective proteins. The asterisk refers to residues that were analyzed by mutagenesis. (B) Wild type (wt) and mutant NFκBp50 RHDs analyzed by Coomassie staining. (C) Radiolabeled RNA probe generated from pBluescript II KS MYB SLR polyU template was incubated with 12.5 ng of recombinant wt or mutant NFκBp50 RHD as indicated and the reactions resolved on a 5% Tris-glycine gel. The black arrow indicates the position of the NFκBp50 RHD-MYB SLR polyU RNA complex; the grey arrow indicates free probe.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122919.g003: The NFκBp50 RHD binds the MYB SLR polyU RNA via a lysine-rich region.(A) Sequence alignment of the RHD of NFκB proteins. Black boxes indicate amino acid identity and grey boxes indicate similarity. The numbers refer to the first position of the segments within the respective proteins. The asterisk refers to residues that were analyzed by mutagenesis. (B) Wild type (wt) and mutant NFκBp50 RHDs analyzed by Coomassie staining. (C) Radiolabeled RNA probe generated from pBluescript II KS MYB SLR polyU template was incubated with 12.5 ng of recombinant wt or mutant NFκBp50 RHD as indicated and the reactions resolved on a 5% Tris-glycine gel. The black arrow indicates the position of the NFκBp50 RHD-MYB SLR polyU RNA complex; the grey arrow indicates free probe.
Mentions: Within the RHD we pinpointed two motifs (aa 139–141; LGI) and (aa 146–148; KKK) that were highly conserved amongst the various human and mouse NFκB family members (Fig 3A). Small clusters of highly basic amino acids rich in Lys residues are features of proteins that bind to RNA, with the positive charge of these residues complementing the negative charged surface of RNA [34]. We therefore sought to determine if the KKK residues contributed to the MYB SLR polyU RNA binding activity of NFκBp50. NFκBp50 RHD mutants were generated in which the Lys residues were substituted singly or collectively with Ala or Asp (Fig 3B). Single substitution of the Lys residues 146, 147 or 148 subtly reduced NFκBp50 RHD binding to MYB SLR polyU RNA (Fig 3C). In contrast, binding of p50 RHD was substantially reduced by substituting all three Lys for Ala or Asp (Fig 3C). The LGI residues were also each substituted for Ala (Fig 3B). Substituting the Leu or Ile residues reduced NFκBp50 binding to MYB SLR polyU RNA probe while substituting the Gly had little effect (Fig 3C).

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