IN-VITRO STUDIES OF THE PRP9-CENTER-DOT-PRP11-CENTER-DOT-PRP21 COMPLEX INDICATE A PATHWAY FOR U2 SMALL NUCLEAR RIBONUCLEOPROTEIN ACTIVATION

Pre-mRNA splicing takes place on a large ribonucleoprotein particle, t he spliceosome which contains the five small nuclear ribonucleoprotein s (snRNPs), U1, U2, U4, U5, and U6. In Saccharomyces cerevisiae the mR NA splicing factors, Prp9, Prp11, and Prp21, are necessary for additio n of the U2 snRNP to the pre-mRNA in an early step of spliceosome asse mbly. This paper describes a study of interactions between these prote ins and their role in spliceosome assembly. The proteins were expresse d in Escherichia coli. Prp9 and Prp11 were purified by metal affinity chromatography. Prp21 was purified using a solubilization/renaturation protocol. We have combined these separately purified proteins and pre sent direct evidence of a Prp9 . Prp11 . Prp21 protein complex that is functional in in vitro splicing assays. Characteristics of this Prp9 . Prp11 . Prp21 complex were further investigated using proteins synth esized in vitro. In addition, we found that Prp9, Prp11, and Prp21 inf luence the structure of the U2 snRNP in a manner that alters the acces sibility of the branch point pairing region of the U2 snRNA to oligonu cleotide- directed RNaseH cleavage. We present a model, based on the d ata presented here and in the accompanying paper, for a combined role of Prp9, Prp11, Prp21, and Prp5 in activating the U2 snRNP for assembl y into the pre-spliceosome.