The RNA branch formed during pre-mRNA splicing occurs at a wide variety of sequences (branch sites] in different mammalian pre-mRNAs. U2 small nuclear ribonucleoprotein (snRNP) binds to the pre-mRNA branch site following the interaction of a protein, U2AF, with the 3' splice site/polypyrimidine tract. Here we show that despite the variability of mammalian branch sites, U2 snRNP has a sequence-specific RNA-binding activity. Thus, RNA branch formation is regulated by two sequence-specific interactions: U2AF with the 3' splice site/polypyrimidine tract, and U2 snRNP with the branch site. The affinity of the branch site for U2 snRNP affects the efficiency of spliceosome assembly and splicing.[Key Words: U2 snRNP; U2AF; branch site; 3' splice site]

We have reconstituted in vitro the four snRNPs known to be involved in pre-mRNA splicing: U1, U2, U5, and U4/6. Reconstitution involves adding either authentic or in vitro-synthesized snRNAs to extracts enriched in snRNP structural polypeptides. The reconstituted snRNPs have the same buoyant density and are immunoprecipitated by the same antibodies as authentic snRNPs. Thus, the polypeptide composition of reconstituted snRNPs is similar, if not identical, to that of authentic snRNPs. We show further that a reconstituted U4/U6 particle is fully functional in forming splicing complexes with pre-mRNA. As is the case for the authentic U4/U6 snRNP, the reconstituted U4 snRNP, but not the U6 snRNA, dissociates from the complex prior to formation of the mature spliceosome. The ability to reconstitute snRNPs and assay their activity in spliceosome formation should provide a powerful approach to study these particles.