Characterization of the nuclear function of vigilin
Date of Completion
Vigilin is a highly conserved mammalian protein that consists of 14 tandem KH domains, which are common nucleic acid binding motifs and are also involved in protein-protein interaction. It is abundant in the cytoplasm but also expressed in the nucleus. In both Drosophila (the DDP1 protein) and S. cerevisiae (Scp160p), knockout of the protein leads to defects in heterochromatin formation. Recently, we identified a nuclear vigilin complex that binds strongly to ADAR-edited RNAs and provided evidence that this complex is involved in heterochromatic gene silencing. To decipher the nuclear role of vigilin in heterochromatin formation, we have now dissected the functional domains of human vigilin by expressing a series of truncated vigilin mutants in HEK293 cells. Using immunoprecipitation we show that vigilin associates in vivo with the histone methyltransferase SUV39H1. Interestingly, the KH13-14 region alone interacts strongly with SUV39H1 in an RNA-independent manner, and expression of this fragment in cells appears to lead to dominant negative effects. Cells transfected with KH13-14 display chromosome segregation defects, similar to those seen when DDP1 is knocked down in insect S2 cells. Further, ChIP analyses with antibodies against trimethylated H3K9 and acetylated histone H4 show that expression of this protein changes the status of pericentric beta satellite and rDNA chromatin from heterochromatic to euchromatic. We speculate that vigilin KH13-14 disrupts the normal function of vigilin by titrating the histone methyltransferase away from chromatin. These data point to the critical role of this RNA-binding protein in the establishment of heterochromatin in mammalian cells. We also studied the mechanism underlying the strong interaction between vigilin and hyperedited RNAs. We found that there is not a specific KH domain that strongly binds I-RNA, instead, more KH domains leads to better binding of vigilin to hyperedited RNA. This provided new evidence that the structure but not the sequence of the RNA is important for the recognizing process by vigilins. ^
Zhou, Jing, "Characterization of the nuclear function of vigilin" (2008). Doctoral Dissertations. AAI3304546.