This activation was dependent on B catenin as siRNA knock down of B catenin caused a significant reduction in the effect of BORIS over expression in the TCF LEF luciferase assay. BORIS associates with polysomes The large amount of RNA including ribosomal RNA, bound to BORIS, suggested that selleck chemical 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 as well as monosomes and polysomes. Inhibitors,Modulators,Libraries A similar sedimentation profile was observed for the ribosomal protein L7.
The majority of BORIS was detected in the light fractions at the top of the gradient, where Inhibitors,Modulators,Libraries it co sediments with the ribosomal proteins S6. The cytoplasmic but non ribosome associated protein, GAPDH, was only detected in the light fractions. Table 1 p values for PANTHER analysis of pathways, molecular function and biological processes of transcripts bound in hNP1 and hNP1 cells differentiated to neurons over 6 days Polysome profiling of HEK293T cells showed a similar sedimentation profile of BORIS to that observed in hNP1 and 6dN cells. Inhibition of translation in HEK293T cells using puromycin, which causes prema ture chain termination and polysomal dissociation shifted BORIS and RPL7 to the first, light fractions.
Furthermore, both RNase A digestion and dissociation of ribosomes into subunits by 30 mM EDTA with the concomitant Brefeldin_A release of mRNA and the 5S ribosomal protein, also shifted the sedimentation of BORIS and to a lesser extent RPL7 towards lighter fractions. Together, these findings suggest that BORIS associates with actively translating ribo somes in these cells. Discussion Here, we provide evidence that BORIS, best known for its role in DNA binding and transcriptional regulation, also binds RNA in vitro and associates with subsets of mRNAs and with translating ribosomes in neural stem cells and young neurons. The ability to bind to both DNA and RNA is not unique to BORIS, and is a feature of certain other zinc finger containing Inhibitors,Modulators,Libraries proteins. The zinc finger domains of BORIS, with which it associ ates with DNA, are almost identical to those in CTCF and Inhibitors,Modulators,Libraries the proteins are reported to share DNA binding sites in the genome. A recent study has suggested that the zinc fingers http://www.selleckchem.com/products/Roscovitine.html in BORIS are needed for both nuclear and nucleolar localisation. It remains to be established whether the zinc finger motifs are important for the RNA binding properties of BORIS, as is the case for TFIIIA, WT1 and certain other proteins.