Antisense Affinity Depletion of RNP Particles: Application to Spliceosomal snRNPs

Complexes composed of RNA and protein play essential roles at multiple levels in the gene expression pathway, including transcription, RNA processing, transport, and translation. In this chapter, we describe a generally applicable system that allows the function of specific RNA-protein complexes to be investigated. Previous strategies used to study the function of small nuclear ribonucleoprotein particles (snRNPs) in vitro have included inhibition with specific antibodies (1 , 2 ), RNase H cleavage of targeted snRNAs in the presence of complementary DNA oligonucleotides (24), and “antisense masking” of specific snRNA sequences with 2′-OMe RNA oligonucleotides (7 –9 ). A potential disadvantage of these methods is that an RNP particle inactivated, but not physically removed from an extract may still retain separate functional domains.