RNA duplexes containing the modified base 2-aminoadenine in place of adenine are stabilized through the formation of three hydrogen bonds in 2-amino A* U base pairs. Antisense 2'-0-alkyloligoribonucleotide probes incorporating 2-aminoadenosine are thus able to efficiently affinity select RNP particles which are otherwise inaccessible. This has allowed the efficient and specific depletion of U5 snRNP from HeLa cell nuclear splicing extracts. U5 snRNP is shown to be essential for spliceosome assembly and for both steps of pre-mRNA splicing. The absence of U5 snRNP prevents the stable association of U4/U6 but not Ul and U2 snRNPs with pre-mRNA. INTRODUCTIONThe splicing of nuclear messenger RNA precursors (pre-mRNA) occurs within a multicomponent structure termed the spliceosome. The major components of the spliceosome belong to the U-class of small nuclear ribonucleoprotein particles (U snRNPs), specifically Ul, U2, U4/U6 and U5 snRNPs. These four snRNPs, together with non-snRNP protein factors, assemble along an ordered pathway to form a functional spliceosome (for recent reviews see 1-4). A combination of affinity selection methods and native gel analyses have shown that the U1, U2, U4/U6 and U5 snRNPs are present in splicing complexes (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). In order to study the roles of the different snRNP particles in the splicing mechanism, a variety of methods have been used to inactivate individual snRNP particles in in vitro splicing extracts. These include inhibition by snRNP-specific antibodies (15-17), site-specific cleavage of snRNA components by RNase H in the presence of complementary DNA oligonucleotides (17-21), and masking specific snRNA domains using antisense 2'-0-methyl oligoribonucleotides (2'-OMe RNA) (22 -24). In Saccharomyces cerevisiae it has also been possible to exploit a genetic approach in which expression of a specific snRNA gene is placed under the control of the inducible p3-gal promoter. This has allowed in vivo depletion of specific snRNP species (25 -27). Using one or more of the above methods, U 1,

Biotinylated 2′‐OMe RNA oligonucleotides complementary to two separate regions of human U2 snRNA have been used as affinity probes to study U2 snRNP‐‐pre‐mRNA interactions. Both oligonucleotides bind specifically and allow highly selective removal of U2 snRNP from HeLa cell nuclear extracts. Pre‐mRNA substrates can also be specifically affinity selected through oligonucleotides binding to U2 snRNP particles in splicing complexes. Stable binding of U2 snRNP to pre‐mRNA is blocked by the pre‐binding of an oligonucleotide to the branch site complementary region of U2 snRNA, but not by an oligonucleotide binding to the 5′ terminus of U2. Both oligonucleotides affinity select the intron product, but not the intron intermediate, when added after spliceosome assembly has taken place. The effect of 2′‐OMe RNA oligonucleotides on splicing complex formation has been used to demonstrate that complexes containing U2 snRNP and unspliced pre‐mRNA are precursors to functional spliceosomes.

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