Characterization of proteins that bind to inosine-containing RNA

Date of Completion

January 2004


Biology, Cell




Double-stranded RNA (dsRNA) in the nucleus triggers at least two distinct biological consequences. First, long dsRNA can be promiscuously edited by a ubiquitously expressed enzyme, Adenosine Deaminase which Acts on dsRNA (ADAR), which deaminates half of the adenosines residues to inosines. These hyperedited RNAs are not exported to the cytoplasm but are retained in the nucleus by binding to a complex containing the inosine-specific RNA binding protein p54 nrb, the splicing factor PSF, and the inner nuclear matrix structural protein matrin 3. Second, dsRNAs can induce gene silencing on heterochromatin, at least some of which appears to be mediated by the RNAi interference (RNAi) machinery. This thesis work provides evidence that ADAR editing might also be involved in this second dsRNA response pathway. This work shows that an abundant and conserved multiple-KH domain protein, vigilin, binds tightly and specifically to inosine-containing RNAs. Immunostaining experiment shows that most vigilin is located in the cytoplasm, where it is associated with the endoplasmic reticulum and polysomes while a fraction of this protein is also in the nucleus, associated with heterochromatin. To investigate the nuclear function of vigilin, we knocked down the homologous protein in Drosophila S2 cells, DDP1. DDP1 deficient cells grow slower, HP1 is mislocated in the nucleus and DNA content is changed compared with control cells. Further biochemical experiments show that nuclear vigilin is in a complex with the Ku heterodimer (Ku70/Ku86) and RNA helicase A (RHA). In presence of RNA, this complex recruits and activates a kinase, the DNA-PKcs, which phosphorlyrates a limited substrates, including HP1α, H2AX and RHA which have been reported to be involved in heterochromatic gene silencing This work points to a heretofore unappreciated role of the RNA editing machinery in dsRNA-mediated gene silencing. This thesis work also include binding studies of inosine-containing RNA binding proteins, p54nrb and vigilin. Both proteins are conserved among species. Here, we show that ability to bind inosine-containing RNAs is also conserved, which suggests that these proteins might be important during evolution. ^