Immunopurification of endogenous RNAs associated with RNA binding proteins In vivo

RNA binding proteins (RBPs) regulate gene expression at several steps from transcription to translation. RBPs can influence both the information content and abundance of RNAs through various regulatory processes, such as pre-mRNA splicing, RNA editing, polyadenylation, RNA stability, and export. The key role of RBPs in the regulation of gene expression is evidenced by many diseases caused by mutations in the RNA sequences bound by RBPs. However, in most cases only few endogenous genes regulated by individual RBPs have been identified. The identification of ribonucleoprotein (RNPs) complexes with which RBPs are associated can yield information on how different RBPs contribute to the regulation of gene expression in cells and tissues. In this section, we describe the immunopurification of mRNPs from tissue culture cells. To systemically determine which mRNAs are associated with individual SR protein splicing factors, SR protein?RNA complexes were purified using a green fluorescent protein (GFP)-affinity tag [1]. The immunopurified mRNAs were analyzed by gene expression microarray and reverse transcription polymerase chain reaction (RT-PCR). Protocols using both uncrosslinked and formaldehyde-crosslinked cell extracts are provided, as well as alternative methods for the microarray as the downstream analysis platform.