Oligonucleotide-Targeted RNase H Protection Analysis of RNA-Protein Complexes

This chapter focuses on the analysis of RNA-protein complexes (RNPs, ribonucleoprotein particles) by oligonucleotide-targeted RNase H digestion, a powerful approach to probe the domain structure of an RNP, both in crude extracts and in purified preparations. RNase H requires DNA-RNA hybrids of at least four basepairs for cleaving the RNA strand (1 ). Therefore, RNase H-mediated cleavage can be directed to a short, specific RNA sequence by hybridization of a complementary DNA oligonucleotide. Oligonucleotide-targeted RNase H cleavage was first used to analyze RNA structures (see , e.g., the analysis of U1 snRNA secondary structure by Lazar and Jacob [2 ] and Rinke et al. [3 ], the characterization of RNA lariat structures [4 ], and the structural probing of a specific mRNA by Hwang et al. [5 ]). The approach has been adopted to RNA-protein complexes, and an important early example is the study of the basepairing potential of the 5′ end of U1 snRNA in the U1 snRNP (6 ). Subsequently, oligonucleotide-targeted RNase H cleavage has been used in various systems to disrupt RNPs for functional studies and in protection assays to map RNP protein-binding domains. Examples include in vitro studies that established snRNA sequences essential for spliceosome assembly and pre-mRNA splicing in the mammalian system and in yeast (see , e.g., refs. 7 –12 ), studies on the assembly of the yeast and mammalian spliceosome (13 ,14 ), investigations on the U7 snRNA function in histone 3′ end processing (e.g., see ref. 15 ), and the analysis of snRNA requirements for trans splicing in trypanosome cells (16 ).