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BORIS/CTCFL is an RNA-binding protein that associates with polysomes.

Ogunkolade BW, Jones TA, Aarum J, Szary J, Owen N, Ottaviani D, Mumin MA, Patel S, Pieri CA, Silver AR, Sheer D - BMC Cell Biol. (2013)

Bottom Line: The majority of the BORIS-associated transcripts are different in the two cell types.Finally, by using polysome profiling we show that BORIS is associated with actively translating ribosomes.We suggest that BORIS is involved in gene expression at both the transcriptional and post-transcriptional levels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Neuroscience and Trauma, Queen Mary University of London, Blizard Institute, Barts and the London School of Medicine and Dentistry, London, E1 2AT, UK. d.sheer@qmul.ac.uk.

ABSTRACT

Background: BORIS (CTCFL), a paralogue of the multifunctional and ubiquitously expressed transcription factor CTCF, is best known for its role in transcriptional regulation. In the nucleus, BORIS is particularly enriched in the nucleolus, a crucial compartment for ribosomal RNA and RNA metabolism. However, little is known about cytoplasmic BORIS, which represents the major pool of BORIS protein.

Results: We show, firstly, that BORIS has a putative nuclear export signal in the C-terminal domain. Furthermore, BORIS associates with mRNA in both neural stem cells and young neurons. The majority of the BORIS-associated transcripts are different in the two cell types. Finally, by using polysome profiling we show that BORIS is associated with actively translating ribosomes.

Conclusion: We have demonstrated the RNA binding properties of cellular BORIS and its association with actively translating ribosomes. We suggest that BORIS is involved in gene expression at both the transcriptional and post-transcriptional levels.

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Related in: MedlinePlus

Western blot analysis of oligo-dT-precipitated RNA-protein complexes. (A) RNA-protein complexes form human neural progenitor cells (hNP1) and hNP1 cells differentiated to neurons over 6 days (6dN) were precipitated with oligo-dT beads analysed by SDS page western blot probed with anti-BORIS antibody (ab18337, Abcam). (B) Immuno-blotting of oligo-dT-RNA bound protein complexes from HEK293T cells transiently expressing BORIS (C3-BORIS) or empty vector (C3-empty). Western blot analysis of Input (2%) and oligo-dT bound protein complex probed with either anti- GFP or anti-BORIS.
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Figure 2: Western blot analysis of oligo-dT-precipitated RNA-protein complexes. (A) RNA-protein complexes form human neural progenitor cells (hNP1) and hNP1 cells differentiated to neurons over 6 days (6dN) were precipitated with oligo-dT beads analysed by SDS page western blot probed with anti-BORIS antibody (ab18337, Abcam). (B) Immuno-blotting of oligo-dT-RNA bound protein complexes from HEK293T cells transiently expressing BORIS (C3-BORIS) or empty vector (C3-empty). Western blot analysis of Input (2%) and oligo-dT bound protein complex probed with either anti- GFP or anti-BORIS.

Mentions: To investigate if BORIS associates with endogenous RNA in hNP1 cells and hNP1 cells differentiated to neurons over 6 days (designated 6dN), we used oligo dT beads to precipitate mRNA from cell lysates and analysed co-precipitated proteins by Western Blot. In both cell types, BORIS was precipitated (Figure 2A), suggesting that the protein associates with mRNA. Similar results were obtained by oligo-dT-precipitation of protein complexes from HEK293T cells transiently expressing GFP-tagged BORIS protein, as detected by both anti- GFP antibodies and anti-BORIS antibodies (Figure 2B). No GFP was precipitated from cell lysates expressing GFP only (Figure 2B).


BORIS/CTCFL is an RNA-binding protein that associates with polysomes.

Ogunkolade BW, Jones TA, Aarum J, Szary J, Owen N, Ottaviani D, Mumin MA, Patel S, Pieri CA, Silver AR, Sheer D - BMC Cell Biol. (2013)

Western blot analysis of oligo-dT-precipitated RNA-protein complexes. (A) RNA-protein complexes form human neural progenitor cells (hNP1) and hNP1 cells differentiated to neurons over 6 days (6dN) were precipitated with oligo-dT beads analysed by SDS page western blot probed with anti-BORIS antibody (ab18337, Abcam). (B) Immuno-blotting of oligo-dT-RNA bound protein complexes from HEK293T cells transiently expressing BORIS (C3-BORIS) or empty vector (C3-empty). Western blot analysis of Input (2%) and oligo-dT bound protein complex probed with either anti- GFP or anti-BORIS.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219345&req=5

Figure 2: Western blot analysis of oligo-dT-precipitated RNA-protein complexes. (A) RNA-protein complexes form human neural progenitor cells (hNP1) and hNP1 cells differentiated to neurons over 6 days (6dN) were precipitated with oligo-dT beads analysed by SDS page western blot probed with anti-BORIS antibody (ab18337, Abcam). (B) Immuno-blotting of oligo-dT-RNA bound protein complexes from HEK293T cells transiently expressing BORIS (C3-BORIS) or empty vector (C3-empty). Western blot analysis of Input (2%) and oligo-dT bound protein complex probed with either anti- GFP or anti-BORIS.
Mentions: To investigate if BORIS associates with endogenous RNA in hNP1 cells and hNP1 cells differentiated to neurons over 6 days (designated 6dN), we used oligo dT beads to precipitate mRNA from cell lysates and analysed co-precipitated proteins by Western Blot. In both cell types, BORIS was precipitated (Figure 2A), suggesting that the protein associates with mRNA. Similar results were obtained by oligo-dT-precipitation of protein complexes from HEK293T cells transiently expressing GFP-tagged BORIS protein, as detected by both anti- GFP antibodies and anti-BORIS antibodies (Figure 2B). No GFP was precipitated from cell lysates expressing GFP only (Figure 2B).

Bottom Line: The majority of the BORIS-associated transcripts are different in the two cell types.Finally, by using polysome profiling we show that BORIS is associated with actively translating ribosomes.We suggest that BORIS is involved in gene expression at both the transcriptional and post-transcriptional levels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Neuroscience and Trauma, Queen Mary University of London, Blizard Institute, Barts and the London School of Medicine and Dentistry, London, E1 2AT, UK. d.sheer@qmul.ac.uk.

ABSTRACT

Background: BORIS (CTCFL), a paralogue of the multifunctional and ubiquitously expressed transcription factor CTCF, is best known for its role in transcriptional regulation. In the nucleus, BORIS is particularly enriched in the nucleolus, a crucial compartment for ribosomal RNA and RNA metabolism. However, little is known about cytoplasmic BORIS, which represents the major pool of BORIS protein.

Results: We show, firstly, that BORIS has a putative nuclear export signal in the C-terminal domain. Furthermore, BORIS associates with mRNA in both neural stem cells and young neurons. The majority of the BORIS-associated transcripts are different in the two cell types. Finally, by using polysome profiling we show that BORIS is associated with actively translating ribosomes.

Conclusion: We have demonstrated the RNA binding properties of cellular BORIS and its association with actively translating ribosomes. We suggest that BORIS is involved in gene expression at both the transcriptional and post-transcriptional levels.

Show MeSH
Related in: MedlinePlus