Small nuclear ribonucleoprotein (snRNP) U2 functions in the splicing of mRNA by recognizing the branch site of unspliced mRNA. The binding of U2 snRNP and other components to pre-mRNA leads to the formation of a stable prespliceosome. In HeLa nuclear extracts, U2 snRNP exists either as a 17S form (under low salt conditions) or a 12S form (at higher salt concentrations). We have recently shown that the purified 17S U2 snRNP contains nine proteins with apparent molecular masses of 35, 53, 60, 66, 92, 110, 120, 150, and 160 kDa in addition to the common snRNP proteins and the U2 proteins A' and B" that are found in the 12S U2 snRNP form.By using antibodies against the PRP9 protein from Saccharomyces cerevisiae (a protein required for the addition of U2 to prespliceosomes in yeast), we have shown that the 60-kDa protein specific to human U2 snRNP particles is structurally related to the yeast PRP9 protein. Interestingly, anti-PRP9 antibodies strongly inhibit prespliceosome formation in HeLa nuclear splicing extracts, resulting in a complete inhibition of the mRNA splicing reaction in vitro. This indicates that the U2 60-kDa protein may also be functionally related to its yeast counterpart PRP9. Most importantly, the addition of purified 17S U2 snRNPs, but not of 12S U2 snRNPs, to HeLa splicing extracts in which the endogeneous U2 snRNPs have been functionally neutralized with anti-PRP9 antibodies fully restores the mRNA-splicing activity of the extracts. These data suggest further that the 17S form is the functionally active form of U2 snRNP in the spliceosome.The catalysis of intron removal from eukaryotic nuclear pre-mRNA molecules requires the activity of four abundant nuclear U small nuclear ribonucleoprotein (snRNP) species Ul, U2, U4/U6, and U5 (for reviews, see refs. 1-3). These assemble with an as yet unestablished number of auxiliary factors and the pre-mRNA substrate into a dynamic RNP complex, termed the spliceosome (for reviews, see refs. 4 and 5). Like the constitutive splicing mechanism, the pathway of spliceosomal assembly has been substantially conserved between yeast and humans (4, 6, 7).As an important early step during spliceosome formation, the U2 snRNP binds to the branch site of the intron (8)(9)(10). This interaction, which involves base pairing between sequences near the 5' end of the U2 snRNA with the branch point sequence (11)(12)(13)(14) depends on the hydrolysis ofATP and leads to the formation of a stable prespliceosomal complex (15-17).The requirements for U2 snRNP addition to the pre-mRNA have been studied to some extent: In Saccharomyces cerevisiae, several lines ofevidence indicate an ATP-independent interaction of the Ul snRNP with the 5' splice site and the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.3'-terminal region of the intron, which commits the premRNA substrate to the splicing reaction, as an essential p...