Proteomic analysis of RNA-dependent chromatin association of nuclear proteins

Various coding and non-coding transcripts are known to associate with chromatin and now there is accumulating evidence that interaction between RNA-binding proteins (RBPs) and RNA molecules regulate not only co-transcriptional mRNA processing, but also other biological processes within the nucleus. Although over a thousand of RBPs have been identified by several mass spectrometry-based methods, it is still unclear which of these RBPs actually associate with chromatin, especially through interaction with RNAs. In addition, biological outcomes of such RBP-RNA-chromatin interactions are yet to be elucidated.\n\nHere we describe a simple proteomics-based method for systematic screening of RBPs that are anchored to chromatin and/or insoluble nuclear substructures by RNA molecules. We used RNase A to release such RBPs from chromatin fraction and analyzed RNase A-solubilized proteins by mass spectrometry. Using this method, we were able to identify 156 RNase A-solubilized proteins of which 144 were known RBPs/RBP candidates. Interestingly, several key players of the non-homologous end-joining (NHEJ) pathway were enriched in RNase A-solubilized fraction and the RNA-mediated chromatin association of these factors appeared to be dependent on transcriptional elongation. Furthermore, some enzymes involved in metabolic pathways were also released from chromatin and/or an insoluble nuclear structure by RNase A treatment. In summary, our methodology is highly versatile and is potentially a useful tool to unravel new biological functions for RBP-RNA-chromatin interactions.