Functional links between the Prp19-associated complex, U4/U6 biogenesis, and spliceosome recycling.

The Prp19-associated complex, consisting of at least eight protein components, is involved in spliceosome activation by specifying the interaction of U5 and U6 with pre-mRNA for their stable association with the spliceosome after U4 dissociation. We show here that yeast cells depleted of one or two of the Prp19-associated components, accumulate the free form of U4. In NTC25-deleted cells, the level of U6 was also reduced. Extracts prepared from NTC25-deleted cells contained neither free U4 nor U6 and were ineffective in spliceosome recycling in the in vitro splicing reaction. Overexpression of U6 partially rescued the temperature-sensitive growth defect and decreased the relative amount of free U4 in NTC25-deleted cells, indicating that the accumulation of free U4 was a consequence of insufficient amounts of U6 snRNA. Extracts prepared from U6-overproducing NTC25-deleted cells containing free-form U6 were capable of spliceosome recycling, suggesting a role of free U6 RNP in spliceosome recycling. Our results demonstrate that in addition to direct participation in spliceosome activation, the Prp19-associated complex has an indirect role in spliceosome recycling through affecting the biogenesis of U4/U6 snRNP in the in vivo splicing reaction.