br Materials and Methods br Results br

Materials and Methods
Results
Discussion We first confirmed that translational repression of target mRNAs by vigilin (Figure 1). It is known that vigilin binds a large number of mRNAs (estimated 700 mRNAs; [12]). We demonstrate that vigilin directly interacts with the pyrimidine-rich element in the CSF-1R mRNA 3′UTR, and requires the full-length 69 nt for stable binding (Figure 2). We show that mutation of the 69 nt CSF-1R element dramatically alters CSF-1R translational efficiency, and obviates vigilin effect on translational repression of CSF-1R (Figure 3). Our data does not rule out a contribution of vigilin on mRNA decay to these experiments on translation, as the mechanisms of mRNA stability and translation are closely coupled. In fact, we have previously shown that vigilin OE can enhance CSF-1R mRNA decay by approximately 1.8-fold, while vigilin silencing had less of an effect on mRNA stability [9]. What is clear collectively is that the primary effect of vigilin is on translation, with a more minor component on mRNA decay. This pyrimidine-rich 69 nt CSF-1R element is a target for binding by proteins other than vigilin. Translational repression and mRNA decay may be reversible by competing CSF-1R mRNA with other RNA binding proteins. We have previously characterized one of them, HuR, with a ABT between vigilin and HuR demonstrated for binding this element, with opposing effects on CSF-1R expression [9], [13], [19]. The binding of vigilin to the pyrimidine-rich sequence significantly decreases CSF-1R expression (Figures 2 and 4). In contrast, the binding of HuR to this pyrimidine rich region increases the CSF-1R expression [13]. The present data indicates that the 69 nt CSF-1R binding element confers net translational repression, and that the repression effect is very significant in the presence of cellular RNA binding proteins, including vigilin and HuR (Figure 3). We have shown that the affinity of vigilin to this pyrimidine-rich region in the CSF-1R mRNA 3′UTR is at least 3-fold stronger than that of HuR in BT20 breast cancer cells by in vitro competition assay [19]. Thus, this specificity of binding of the 69 nt pyrimidine-rich region to vigilin, may explain why disruption of the pyrimidine-rich sequence appears to more significantly affect vigilin-specific actions, rather than those of HuR. We then explored the potential roles of vigilin, along with other proteins which have affinity for the 69 nt CSF-1R element, in breast cancer cells. The sequestration of these proteins by excess 69 nt pyrimidine-rich element increases CSF-1R levels (Figures 2 and 4), and as a result, increases the degree of adhesion, motility, and invasion of breast cancer cells (Figure 4). It is notable that the degree of effect on up-regulation of CSF-1R is similar whether the terminal 578 nt CSF-1R mRNA 3′UTR containing the pyrimidine-rich sequence is overexpressed (Figure 4), or just the 69 nt pyrimidine-rich sequence (Figure 2). This underlies the impact of this small pyrimidine-rich region on post-transcriptional regulation of CSF-1R mRNA, as well as on breast cancer cellular behavior in vitro.