Enriched With Previously Uncharacterized RNA-protein Interactions In PNC

The cell nucleus is a highly organized structure. It is arranged into distinct membrane-lacking nuclear bodies. This compartmentalization is thought to represent the most efficient organization of essential molecular complexes. Our research focuses on one such nuclear body, the perinucleolar compartment (PNC).The PNC is a subnuclear structure that is associated with malignant transformation. High PNC prevalence (number of cells containing one or more PNC) correlates with metastatic capacity and indicates poor prognosis in cancer models. To determine the functional association between the PNC and malignancy, we investigated the molecular complexes that are associated with the PNC. The PNC is enriched with pol III transcribed RNA (ie. MRP, RNase P) and pol II RNA binding proteins (ie. PTB, CUGBP).To explore the protein-RNA interactions we used pull down and immunoprecipitation experiments and observed that MRP RNA, PTB and CUGBP precipitate each other. Evaluation of the components’distribution in the nucleus using the high resolution (OMX) system demonstrates that MRP RNA colocalizes with PTB and CUGBP in a reticulated network fashion. The enrichment of MRP RNA in the PNC appears to be independent of previously characterized interactions with the hTERT protein or MRP RNP complex, as the protein subunits of these complexes do not co-enrich with the PNC. Furthermore, PTB and CUGBP are able to recruit each other when tethered at a LacO site inserted into the genome.PNC-enriching PTB and CUGBP behave differently than their nucleoplasmic counterparts, with regards to their redistribution pattern in the absence of pre-mRNA synthesis and their recovery dynamics after photobleaching. Glycerol gradient analysis demonstrates that the MRP RNA-PTB-CUGBP interactions are specific to the bottom fraction, and the complex is large in size. Mass spectrometry has identified a number of additional potential protein components. Furthermore, OMX microscopy of BRU labeled cells shows that the complex intertwines with newly synthesized RNA.These data together suggest that MRP RNA is in a novel RNA-protein complex that nucleates at the PNC and may have a novel function in the regulation of newly synthesized RNA. These results provide a better understanding of the structure and function of the PNC.