SLU7 AND A NOVEL ACTIVITY, SSF1, ACT DURING THE PRPL6-DEPENDENT STEP OF YEAST PRE-MESSENGER-RNA SPLICING

Understanding the mechanism of pre-mRNA splicing requires the characte rization of all components involved. In the present study, we used the genetically and biochemically defined yeast PRP16 protein as a point of departure for the identification of additional factors required for the second catalytic step in vitro. We isolated by glycerol gradient sedimentation spliceosomes that were formed in yeast extracts depleted of PRP16. This procedure separated the spliceosomal complexes contain ing lariat intermediate and exon 1 from free proteins present in the w hole-cell yeast extract. We then supplemented these spliceosomes with purified proteins or yeast extract fractions as a functional assay for second-step splicing factors, We show that SLU7 protein and a novel a ctivity that we named SSF1 (second-step factor 1) were required in con cert with PRP16 to promote progression through the second catalytic st ep of splicing. Taking advantage of a differential ATP requirement for PRP16 and SLU7 function, we show that SLU7 can act after PRP16 in the splicing pathway.