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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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BORIS binds directly to RNA in a nucleotide-specific manner. (A) SDS-PAGE western blot analysis of the purification of Halo tagged BORIS from transfected HEK293T cells. (B)In vitro binding assay using biotinylated RNA and DNA homopolymers and purified recombinant BORIS from (A). BORIS showed association with poly(rG) and poly(rU), but not with poly(rA) or poly(rC). BORIS also showed specific affinity for poly(dT), poly(dG) and poly(dC) DNA while no binding was observed to poly(dA) or the streptavidin beads alone (No oligo, lane 1). (C) Venn diagram showing the number of identified BORIS associated transcripts in hNP1 and hNP1 cells differentiated to neurons over 6 days (6dN) as well as the number of transcripts associated with BORIS in both cell types. RT-qPCR confirmation of BORIS associated transcripts in hNP1 (D) and 6dN cells (E). Data in (D) and (E) represent 2 technical replicates ± SD.
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Figure 4: BORIS binds directly to RNA in a nucleotide-specific manner. (A) SDS-PAGE western blot analysis of the purification of Halo tagged BORIS from transfected HEK293T cells. (B)In vitro binding assay using biotinylated RNA and DNA homopolymers and purified recombinant BORIS from (A). BORIS showed association with poly(rG) and poly(rU), but not with poly(rA) or poly(rC). BORIS also showed specific affinity for poly(dT), poly(dG) and poly(dC) DNA while no binding was observed to poly(dA) or the streptavidin beads alone (No oligo, lane 1). (C) Venn diagram showing the number of identified BORIS associated transcripts in hNP1 and hNP1 cells differentiated to neurons over 6 days (6dN) as well as the number of transcripts associated with BORIS in both cell types. RT-qPCR confirmation of BORIS associated transcripts in hNP1 (D) and 6dN cells (E). Data in (D) and (E) represent 2 technical replicates ± SD.

Mentions: Next, to determine whether BORIS binds directly to RNA, a series of 20 mer RNA and DNA homopolymers with 3’ Biotin-TEG was utilised in an in vitro binding assay. Recombinant BORIS was purified from HEK293T cells (Figure 4A) and assayed for its ability to bind to the biotin-coupled homopolymers. As expected, we found that BORIS associates with the DNA homopolymers poly(dT), poly(dG) and poly(dC) [30]. In addition, BORIS also bound to poly(rG) and, to a lesser extent, to poly(rU) RNAs, while no binding was observed to polymers of rC or rA or to the streptavidin beads alone (Figure 4B). These experiments suggest that BORIS can interact directly with RNA.


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)

BORIS binds directly to RNA in a nucleotide-specific manner. (A) SDS-PAGE western blot analysis of the purification of Halo tagged BORIS from transfected HEK293T cells. (B)In vitro binding assay using biotinylated RNA and DNA homopolymers and purified recombinant BORIS from (A). BORIS showed association with poly(rG) and poly(rU), but not with poly(rA) or poly(rC). BORIS also showed specific affinity for poly(dT), poly(dG) and poly(dC) DNA while no binding was observed to poly(dA) or the streptavidin beads alone (No oligo, lane 1). (C) Venn diagram showing the number of identified BORIS associated transcripts in hNP1 and hNP1 cells differentiated to neurons over 6 days (6dN) as well as the number of transcripts associated with BORIS in both cell types. RT-qPCR confirmation of BORIS associated transcripts in hNP1 (D) and 6dN cells (E). Data in (D) and (E) represent 2 technical replicates ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: BORIS binds directly to RNA in a nucleotide-specific manner. (A) SDS-PAGE western blot analysis of the purification of Halo tagged BORIS from transfected HEK293T cells. (B)In vitro binding assay using biotinylated RNA and DNA homopolymers and purified recombinant BORIS from (A). BORIS showed association with poly(rG) and poly(rU), but not with poly(rA) or poly(rC). BORIS also showed specific affinity for poly(dT), poly(dG) and poly(dC) DNA while no binding was observed to poly(dA) or the streptavidin beads alone (No oligo, lane 1). (C) Venn diagram showing the number of identified BORIS associated transcripts in hNP1 and hNP1 cells differentiated to neurons over 6 days (6dN) as well as the number of transcripts associated with BORIS in both cell types. RT-qPCR confirmation of BORIS associated transcripts in hNP1 (D) and 6dN cells (E). Data in (D) and (E) represent 2 technical replicates ± SD.
Mentions: Next, to determine whether BORIS binds directly to RNA, a series of 20 mer RNA and DNA homopolymers with 3’ Biotin-TEG was utilised in an in vitro binding assay. Recombinant BORIS was purified from HEK293T cells (Figure 4A) and assayed for its ability to bind to the biotin-coupled homopolymers. As expected, we found that BORIS associates with the DNA homopolymers poly(dT), poly(dG) and poly(dC) [30]. In addition, BORIS also bound to poly(rG) and, to a lesser extent, to poly(rU) RNAs, while no binding was observed to polymers of rC or rA or to the streptavidin beads alone (Figure 4B). These experiments suggest that BORIS can interact directly with RNA.

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