Interactions between Nova and brPTB Proteins in Alternative Splicing and Nuclear Localization
The Nova paraneoplastic antigens are neuron-specific RNA-binding proteins that are essential for neuronal viability and participate in the control of alternative splicing. In this study, the yeast two-hybrid system is used to isolate Nova-1nteracting proteins. A novel RNA-binding protein named brPTB is identified, that is closely related to the polypyrimidine tract-binding protein (PTB) and is ennched in the brain at the mRNA and protein levels. brPTB interacts with Nova proteins in vitro and in vivo. Splicing assays in kidney epithelial cell lines show that brPTB inhibits the effect of Noval in the inclusion of alternatively spliced exons in two target pre-mRNAs: glycine receptor oc2 subunit (GlyRa2) and GABAA receptor y2 subunit (GABAARy2). Furthermore, in the case of GlyRoc2, brPTB binds to a site adjacent to Noval in a 90 nucleotide fragment of intronic RNA upstream of the alternatively spliced exon, but with an affinity more than 10-fold lower than Noval. When the brPTB site is mutated, binding is abolished and the inhibitory effect on Noval-dependent exon inclusion disappears. In addition, it is shown that the inhibitory effect of brPTB on Noval splicing does not occur in neuronal cells. These results suggest that brPTB is a tissue-restricted RNA-binding protein that specifically interacts with Nova and inhibits its ability to activate exon selection. Nova proteins localize in distinct nuclear foci, but fail to co-localize with any of the known proteins that occupy sub-nuclear structural domains. Both endogenous and transfected brPTB and Nova proteins co-localize in these foci in neuroblastoma cell lines but not in non-neuronal cells. A model is proposed whereby alternative splicing and nuclear localization mediated by Nova and brPTB are linked as the consequence of their protein interaction in specific cell types.
Nova proteins, alternative splicing, brPTB, RNA-binding, neuronal specificity, protein interaction, Life Sciences
Networked Digital Library of Theses and Dissertations