The PNC is present almost exclusively in transformed cells and is rarely found in normal cells. The PNC is enriched with small RNAs newly transcribed by pol III and a group of proteins that have been shown to be involved in the processing of pol II transcripts. Colocalization of the RNA and proteins in the PNC suggests novel interactions between them and changes in these interactions in tumor cells may play a role in advancing transformation. Using RNase MRP RNA, that is concentrated in the PNC, as an example, we will (1) identify a novel MRP-RNA containing complex and its components, and (2) isolate and describe molecular interactions that are required for the formation of the novel MRP RNA-containing complex. A series of immunoprecipitation and pull-down assays will be performed in parallel together with proteomic analysis to identify the protein components of the novel complex. A battery of GST-pull down assays and mutational analyses will be used to classify and map interactions within the complex. Lastly, RNAi and/or further mutagenesis will be used to knock-down proteins associated with the novel complex to determine whether they are required for novel complex assembly. ? ? ?
