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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

Polysome profiling of BORIS. (A) Polysome profiles of cell lysates from hNP1 and hNP1 cells differentiated to neurons over 6 days (6dN) on a 10-50% sucrose gradient. Lighter particles at the top of the gradient are shown on the left and heavier fractions are shown on the right. Equal volumes of each fraction were analysed by SDS-PAGE and probed with indicated antibodies. S6 and RPL7 are ribosome-associated proteins while GAPDH is not. (B) Polysome profiling of HEK293T cell lysates, detecting endogenous BORIS, after disruption of polysomes by puromycin (top panel), 30 mM EDTA (middle panel) or following RNAse A digestion (lower panel).
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Figure 7: Polysome profiling of BORIS. (A) Polysome profiles of cell lysates from hNP1 and hNP1 cells differentiated to neurons over 6 days (6dN) on a 10-50% sucrose gradient. Lighter particles at the top of the gradient are shown on the left and heavier fractions are shown on the right. Equal volumes of each fraction were analysed by SDS-PAGE and probed with indicated antibodies. S6 and RPL7 are ribosome-associated proteins while GAPDH is not. (B) Polysome profiling of HEK293T cell lysates, detecting endogenous BORIS, after disruption of polysomes by puromycin (top panel), 30 mM EDTA (middle panel) or following RNAse A digestion (lower panel).

Mentions: The large amount of RNA including ribosomal RNA, bound to BORIS, suggested that BORIS interacts with the translational machinery. To investigate this directly, we performed polysome profiling on cell extracts prepared from hNP1 and 6dN cells and analysed the distribution of BORIS in the resulting gradients by Western blotting. Consistent with a ribosomal association, BORIS was present throughout the gradient, co-sedimenting with all ribosomal subunits (40S and 60S) as well as monosomes (80S) and polysomes (Figure 7A). A similar sedimentation profile was observed for the ribosomal protein L7 (RPL7). The majority of BORIS was detected in the light fractions at the top of the gradient, where it co-sediments with the ribosomal proteins S6. The cytoplasmic but non-ribosome associated protein, GAPDH, was only detected in the light fractions.


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)

Polysome profiling of BORIS. (A) Polysome profiles of cell lysates from hNP1 and hNP1 cells differentiated to neurons over 6 days (6dN) on a 10-50% sucrose gradient. Lighter particles at the top of the gradient are shown on the left and heavier fractions are shown on the right. Equal volumes of each fraction were analysed by SDS-PAGE and probed with indicated antibodies. S6 and RPL7 are ribosome-associated proteins while GAPDH is not. (B) Polysome profiling of HEK293T cell lysates, detecting endogenous BORIS, after disruption of polysomes by puromycin (top panel), 30 mM EDTA (middle panel) or following RNAse A digestion (lower panel).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Polysome profiling of BORIS. (A) Polysome profiles of cell lysates from hNP1 and hNP1 cells differentiated to neurons over 6 days (6dN) on a 10-50% sucrose gradient. Lighter particles at the top of the gradient are shown on the left and heavier fractions are shown on the right. Equal volumes of each fraction were analysed by SDS-PAGE and probed with indicated antibodies. S6 and RPL7 are ribosome-associated proteins while GAPDH is not. (B) Polysome profiling of HEK293T cell lysates, detecting endogenous BORIS, after disruption of polysomes by puromycin (top panel), 30 mM EDTA (middle panel) or following RNAse A digestion (lower panel).
Mentions: The large amount of RNA including ribosomal RNA, bound to BORIS, suggested that BORIS interacts with the translational machinery. To investigate this directly, we performed polysome profiling on cell extracts prepared from hNP1 and 6dN cells and analysed the distribution of BORIS in the resulting gradients by Western blotting. Consistent with a ribosomal association, BORIS was present throughout the gradient, co-sedimenting with all ribosomal subunits (40S and 60S) as well as monosomes (80S) and polysomes (Figure 7A). A similar sedimentation profile was observed for the ribosomal protein L7 (RPL7). The majority of BORIS was detected in the light fractions at the top of the gradient, where it co-sediments with the ribosomal proteins S6. The cytoplasmic but non-ribosome associated protein, GAPDH, was only detected in the light fractions.

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