Abstract 1089: Dissecting the potential distinct role/s of the four RNA binding domains in a phosphoprotein nucleolin using bioinformatics tools

Abstract

Abstract Nucleolin is an abundant RNA binding protein within the nucleolus containing four copies of a consensus RNA-binding domain (RBD1-4). Two of these contiguous RNA-binding domains (RBD12) are sufficient to bind to a variety of RNAs (e.g. rRNA, mRNA, ncRNA) through their highly conserved RNP-1/2 motifs. Nucleolin controls gene expression either post-transcriptionally through mRNA stability or by post-translational regulation, targeting mRNAs with ARE (AU-rich elements), NRE (nucleolin recognition elements) and/or G-rich regions. Nucleolin's RNA binding functions are directly implicated in various pathological conditions e.g. Alzheimer's disease, cancer and viral infection. Interestingly, nucleolin RNA binding properties and phosphorylation pattern change during the cellular response to stress suggesting possible collaboration in regulating gene expression. However, most RNA binding properties of nucleolin studied to date exclude its highly acidic N-terminus phosphorylation domain. Importantly, NMR structures are only available for RBD12 although RBD1-4 of nucleolin is required to interact with the evolutionary conserved motif (ECM) on pre-RNAs, which have important role/s in RNA processing. Our work in accordance with others has shown that the N-terminus domain plays a vital role in cell proliferation and therefore has potential to influence RNA-binding properties of nucleolin to regulate gene expression. Here we use bioinformatics tools to elucidate the RNA binding functions of all four RBDs of nucleolin individually and in various combinations. Using I-TASSER, a tertiary structure prediction algorithm, and the available structural data for RBD12, we first predicted models for RBD3 and 4.These models displayed nearly perfect superimposition of RBD34 with RBD12 where RBD3/2 and RBD4/1 align. Our central hypothesis is that nucleolin RBD3/4 domains can replace RBD1/2 domains in binding to RNAs. To test our hypothesis we have generated a variety of theoretical constructs of RBD1-4 with mutations in the RNP1/2 conserved sites of individual RBDs. We present the computational analyses of these constructs to predict the role/s of RBD3/4. We further predict any role/s imparted by N-terminus phosphorylation and/or the C-terminus GAR (Gly-Arg rich) domains in regulating RNA binding by nucleolin. This bioinformatics-based knowledge will provide useful insights about nucleolin-RNA interactions that can be framed into rationally designed experiments. Citation Format: Ruchama Chaya Steinberg, Shaneen Singh, Anjana D. Saxena. Dissecting the potential distinct role/s of the four RNA binding domains in a phosphoprotein nucleolin using bioinformatics tools. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1089. doi:10.1158/1538-7445.AM2015-1089