This chapter describes methods for RNA–protein cross-linking in living cells as a means of identification of RNA-binding proteins in vivo. The chapter focuses on heterogeneous nuclear RNA (hnRNA)- and messenger RNA (mRNA)-binding proteins. The most stringent definition of genuine ribonucleoproteins (RNPs) is that these are proteins, which are bound directly to the RNA of interest in the living cell. UV cross-linking of RNP complexes takes advantage of the fact that UV light of sufficient intensity generates highly reactive species of RNA, which react virtually indiscriminately with molecules, including proteins, with which the RNA is in stable, direct contact. The cross-linked proteins can be released from the RNA by exhaustive RNase digestion and can be analyzed by gel-electrophoretic techniques. This method provides a powerful tool for the identification of bona fide RNP proteins. The fractionation of cells into nuclear and cytoplasmic components prior to the chromatographic step allows the distinction between hnRNA-containing RNP complexes (hnRNPs), from the nuclear fraction, and mRNAcontaining RNP complexes (mRNPs), from the cytoplasmic fraction.
ASJC Scopus subject areas
- Molecular Biology