Suppressors of a cold-sensitive mutation in yeast U4 RNA define five domains in the splicing factor Prp8 that influence spliceosome activation

The highly conserved splicing factor Prp8 has been implicated in multiple s tages of the splicing reaction. However, assignment of a specific function to any part of the 280-kD U5 snRNP protein has been difficult, in part beca use Prp8 lacks recognizable functional or structural motifs. We have used a large-scale screen for Saccharomyces cerevisiae PRP8 alleles that suppress the cold sensitivity caused by U4-cs1, a mutant U4 RNA that blocks U4/U6 u nwinding, to identity with high resolution five distinct regions of PRP8 in volved in the control of spliceosome activation. Genetic interactions betwe en two of these regions reveal a potential long-range intramolecular fold. Identification of a yeast two-hybrid inter action, together with previously reported results, implicates two other regions in direct and indirect cont acts to die U1 snRNP. In contrast to the suppressor mutations in PRP8, loss -of-function mutations in the genes for two other splicing factors implicat ed in U4/U6 unwinding, Prp44 (Brr2/Rss1/Slt22/Snu246) and Prp24, show synth etic enhancement with U4-cs1. On the basis of these results we propose a mo del in which allosteric changes in Prp8 initiate spliceosome activation by (1) disrupting contacts between the U1 snRNP and the U4/U6-U5 tri-snRNP and (2) orchestrating the activities of Prp44 and Prp24.