Identification of RNA–Protein Contacts within Functional Ribonucleoprotein Complexes by RNA Site-Specific Labeling and UV Crosslinking

Abstract

A variety of cellular processes are carried out by highly complex ribonucleoprotein (RNP) particles in which multiple RNA–RNA, RNA–protein, and protein–protein interactions occur. The spliceosome, which executes the nuclear pre-mRNA splicing reaction, is a particularly striking example of a complex RNP, containing a minimum of 50 distinct protein components as well as five small nuclear RNAs. In order to identify which among the numerous proteins may play critical roles in the splicing reaction, we have assembled spliceosomal complexes on pre-mRNA containing a single32P-labeled nucleotide, isolated the complexes by gel filtration, and then carried out UV crosslinking. The combination of these three methods has allowed the identification of proteins that crosslink to critical sequence elements during each stage in spliceosome assembly. These methods should be generally applicable to the analysis of RNP complexes assembledin vitro.