Functional dissection of molecular architectures formed by cancer-related noncoding RNAs

Abstract

Recent studies have shown that RNAs act as active molecules in the regulation of various cellular metabolism processes and plays a key role in cancer development or other disease progression. Their biological roles depend on the three-dimensional structures, although specific sequences have been shown to be crucial for various functions. Therefore, analysis of the RNA conformation in cells is essential for understanding their roles. First, we devised a novel tool for detecting and analyzing RNA conformation by anti-RNA antibody. We have developed an efficient strategy for panning and affinity maturation of human monoclonal antibodies binding to RNA from a na"ive antigen binding fragment (Fab) combinatorial phage library, using brain cytoplasmic 200 (BC200) RNA as the antigen. BC200 RNA, a neuron-specific noncoding RNA, is implicated in the inhibition of local synaptodendritic protein synthesis in human cells, and shown to be expressed at higher levels in invasive carcinomas than benign tumors of the breast. Using panning and affinity maturation, we identified MabBC200-A3 as the optimal binder, which interacted specifically with two regions (residues 76 to 85 and 96 to 104) of BC200 RNA with a dissociation constant of ~7 nM. Expression of BC200 RNA in various breast cancer cell lines was further examined using conventional hybridization and immunoanalysis with MabBC200-A3. When total cellular RNAs purified from cells were subjected to hybridization, the signals varied significantly among the cell types. Similar variability of BC200 levels among cell lines was observed upon immunoanalysis with the antibody. Furthermore, the antibody was able to discriminate BC200 RNA from homologous 7SL RNA, supporting its utility as a specific probe for the RNA. Intriguingly, however, when permeabilized cells were used instead of purified total cellular RNA, the amounts of antibody-recognizable BC200 RNA were different, indicating that BC200 RNA exists as at least two distinct cellular forms (antibody-recognizable and nonrecognizable), depending on the structural integrity of the antigenic RNA motif and its accessibility to the antibody. Our data clearly demonstrate the value of the anti-RNA antibody as a novel tool for investigating RNA conformation, which cannot be achieved with hybridization. Second, we analyzed the structural elements of steroid receptor RNA activator (SRA) RNA and suggested a new RNA inhibitor on estrogen receptor alpha (ER $\alpha$) signaling in breast cancers. SRA is an RNA regulator that coactivates steroid receptor-mediated transcription. It is a putative coactivator in the estrogen receptor (ER) signaling pathway. It functions as an RNA molecule itself, but its isoform can encode a protein. SRA levels are elevated in the majority of tumors. Regulatory actions of noncoding SRA core RNA vary depending on the type of estrogen receptors, the presence or absence of ligand, and even reporter elements. Recently it was reported that SRA RNA can be dissected into four domains based on various probing experiments. In this study, we examined the contribution of domains or helices to its coactivation ability to understand the role of structural elements of SRA RNA essential for the RNA function. We showed that domain III played a crucial role in SRA RNA. Furthermore the domain specifically inhibits ER $\alpha$ -dependent transactivation as a competitive inhibitor by interfering with the interaction between coactivators and SRA RNA. In particular four helices, H15 to H18 among nine helices of the domain III showing significantly high sequence conservation across vertebrates also inhibited the ER $\alpha$ signaling pathway. Taken together, the results gave an insight into structural basis for a role of SRA RNA with the estrogen and ER complex and proposed a new target candidate on RNA therapeutics in breast cancers. In summary, this thesis proposed a novel tool for recognizing RNA conformation and dissecting RNA structural motifs. Also we investigated the contribution of structural elements of SRA RNA in breast tumors. In that molecular architecture plays a key role in understanding the mechanism of many functional RNAs, these studies of functional dissection can pave the way for gaining extensive insights on RNA field as well as application to develop new diagnostic and therapeutic approaches for RNA-related diseases.