Functional Analysis of the 15 KH Domain Human Nucleic Acid Binding Protein, Vigilin
Vigilin/Scp160p/DDP1 is a ubiquitous protein containing 15 related, but nonidentical K homology (KH) nucleic acid binding domains. Despite its high level of conservation among species, the precise function(s) of vigilin remain unclear. Vigilin was identified in our lab as the trans-acting factor which binds to the 3' untranslated region of vitellogenin mRNA following estrogen induction. By binding to this region of vitellogenin mRNA, vigilin protects the mRNA from cleavage by the endonuclease PMR-1 and causes the message to be stabilized. Given its identification in various systems by many laboratories, my thesis research has been to begin to elucidate the function of vigilin. Initially, I examined the binding properties of vigilin to two nucleic acid targets: vitellogenin mRNA and a single-stranded satellite sequence found in Drosophila for which vigilin had been reported to have a high affinity. While this data and previous data obtained in our lab have revealed much about vigilin's preferred nucleic acid targets, it told us little about the role of the protein in the cell. To determine what occurs when vigilin levels are reduced, I used RNA interference to knockdown vigilin mRNA levels in HeLa cells. The effective reduction of vigilin levels in cell culture allowed me to test, for the first time, the existing hypotheses that vigilin is important in chromosome partitioning during mitosis, control of mRNA stability, and translation. Furthermore, I was able to use this system to demonstrate that vigilin is essential for HeLa cell viability. In a further effort to identify vigilin's cellular functions, I attempted to identify vigilin's protein interaction partners and mRNA targets. These two methods proved to be technically challenging. I therefore combined microarray analysis with RNA interference mediated reduction in vigilin levels to carry out expression profiling. These experiments allowed me to identify many genes that were up and down-regulated over a time course following vigilin knockdown. Furthermore, I have been able to identify classes of mRNAs and pathways that seem to be affected by the reduction in vigilin levels. The challenge of dissecting these pathways with regard to the importance of vigilin still remains.
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign