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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

HIV-Tat induces MYB expression and transcriptional elongation.(A) The RNA stem loop structure of the HIV TAR [26]. Sequence alignment of the TAR binding region of HIV-1 Tat with the RHD of NFκBp50. Black boxes indicate amino acid identity and grey boxes indicate similarity. The Tat RNA binding domain (ARM motif) is indicated by the black line. The numbers refer to the first position of the segments within the respective proteins. The asterisk refers to NFκBp50 residues that were analyzed by mutagenesis. (B) The genomic arrangement of the MYB locus. The anti-sense RNA probes, the location of the SLR region within intron 1 and the location of RT-PCR primers used to examine RNA elongation across this region by nuclear run-on transcription are shown. (C) Transcription of the MYB gene in 293 cells as assessed by nuclear run-on transcription. Nuclear run on assays were performed as described [20, 21]. Transcriptional activity was normalized to GAPDH signal and the steady state rate of transcription for each transcript length. Relative transcription between untransfected cells and cell transfected with Tat is shown. Densitometric analysis of the radioactivity bound to the filters was performed using Imagequant software and represents the mean values obtained from duplicate filters. (D) Nuclear RNA was isolated from 293 cells transfected with 100 ng pCMVTat (72) and subjected to RT-PCR to detect intron 1 RNA pre- and post-MYB SLR attenuator region.
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pone.0122919.g006: HIV-Tat induces MYB expression and transcriptional elongation.(A) The RNA stem loop structure of the HIV TAR [26]. Sequence alignment of the TAR binding region of HIV-1 Tat with the RHD of NFκBp50. Black boxes indicate amino acid identity and grey boxes indicate similarity. The Tat RNA binding domain (ARM motif) is indicated by the black line. The numbers refer to the first position of the segments within the respective proteins. The asterisk refers to NFκBp50 residues that were analyzed by mutagenesis. (B) The genomic arrangement of the MYB locus. The anti-sense RNA probes, the location of the SLR region within intron 1 and the location of RT-PCR primers used to examine RNA elongation across this region by nuclear run-on transcription are shown. (C) Transcription of the MYB gene in 293 cells as assessed by nuclear run-on transcription. Nuclear run on assays were performed as described [20, 21]. Transcriptional activity was normalized to GAPDH signal and the steady state rate of transcription for each transcript length. Relative transcription between untransfected cells and cell transfected with Tat is shown. Densitometric analysis of the radioactivity bound to the filters was performed using Imagequant software and represents the mean values obtained from duplicate filters. (D) Nuclear RNA was isolated from 293 cells transfected with 100 ng pCMVTat (72) and subjected to RT-PCR to detect intron 1 RNA pre- and post-MYB SLR attenuator region.

Mentions: Comparative sequence alignment revealed that residues (aa118-157) of the NFκBp50 RHD were similar to a highly conserved basic region of Tat (aa 48–57), which functions as an RNA binding domain (Arg rich motif; ARM) [26, 38] (Fig 6A). The NFκBp50 RHD shares two Lys residues (KK:aa 146–147) with the ARM of Tat that are important for TAR RNA binding [39]. A third Lys residue (K:aa 148) was a conserved change. Upstream of the ARM, Tat and NFκBp50 also shared Leu, Gly and Ile residues (LGI: aa 139–141) (Fig 6A). This region of Tat is required for its transactivation function and is part of a sequence required for interaction with CyclinT1 [40, 41].


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)

HIV-Tat induces MYB expression and transcriptional elongation.(A) The RNA stem loop structure of the HIV TAR [26]. Sequence alignment of the TAR binding region of HIV-1 Tat with the RHD of NFκBp50. Black boxes indicate amino acid identity and grey boxes indicate similarity. The Tat RNA binding domain (ARM motif) is indicated by the black line. The numbers refer to the first position of the segments within the respective proteins. The asterisk refers to NFκBp50 residues that were analyzed by mutagenesis. (B) The genomic arrangement of the MYB locus. The anti-sense RNA probes, the location of the SLR region within intron 1 and the location of RT-PCR primers used to examine RNA elongation across this region by nuclear run-on transcription are shown. (C) Transcription of the MYB gene in 293 cells as assessed by nuclear run-on transcription. Nuclear run on assays were performed as described [20, 21]. Transcriptional activity was normalized to GAPDH signal and the steady state rate of transcription for each transcript length. Relative transcription between untransfected cells and cell transfected with Tat is shown. Densitometric analysis of the radioactivity bound to the filters was performed using Imagequant software and represents the mean values obtained from duplicate filters. (D) Nuclear RNA was isolated from 293 cells transfected with 100 ng pCMVTat (72) and subjected to RT-PCR to detect intron 1 RNA pre- and post-MYB SLR attenuator region.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122919.g006: HIV-Tat induces MYB expression and transcriptional elongation.(A) The RNA stem loop structure of the HIV TAR [26]. Sequence alignment of the TAR binding region of HIV-1 Tat with the RHD of NFκBp50. Black boxes indicate amino acid identity and grey boxes indicate similarity. The Tat RNA binding domain (ARM motif) is indicated by the black line. The numbers refer to the first position of the segments within the respective proteins. The asterisk refers to NFκBp50 residues that were analyzed by mutagenesis. (B) The genomic arrangement of the MYB locus. The anti-sense RNA probes, the location of the SLR region within intron 1 and the location of RT-PCR primers used to examine RNA elongation across this region by nuclear run-on transcription are shown. (C) Transcription of the MYB gene in 293 cells as assessed by nuclear run-on transcription. Nuclear run on assays were performed as described [20, 21]. Transcriptional activity was normalized to GAPDH signal and the steady state rate of transcription for each transcript length. Relative transcription between untransfected cells and cell transfected with Tat is shown. Densitometric analysis of the radioactivity bound to the filters was performed using Imagequant software and represents the mean values obtained from duplicate filters. (D) Nuclear RNA was isolated from 293 cells transfected with 100 ng pCMVTat (72) and subjected to RT-PCR to detect intron 1 RNA pre- and post-MYB SLR attenuator region.
Mentions: Comparative sequence alignment revealed that residues (aa118-157) of the NFκBp50 RHD were similar to a highly conserved basic region of Tat (aa 48–57), which functions as an RNA binding domain (Arg rich motif; ARM) [26, 38] (Fig 6A). The NFκBp50 RHD shares two Lys residues (KK:aa 146–147) with the ARM of Tat that are important for TAR RNA binding [39]. A third Lys residue (K:aa 148) was a conserved change. Upstream of the ARM, Tat and NFκBp50 also shared Leu, Gly and Ile residues (LGI: aa 139–141) (Fig 6A). This region of Tat is required for its transactivation function and is part of a sequence required for interaction with CyclinT1 [40, 41].

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