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The UAP56 gene has been shown to be required for prespliceosome assembly in mammals. We report here the isolation of theSchizosaccharomyces pombe ortholog of this gene by heterologous complementation of a combinedPRP40HA ₃ /nam8Δ defect in budding yeast. The Saccharomyces cerevisiae ortholog,YDL084w/SUB2, is also able to suppress this defect. We show that SUB2 is involved in splicing in vivo as well as in vitro. Sub2 defective extracts form a stalled intermediate that contains U2snRNP and can be chased into functional spliceosomes. Our experiments also suggest a role for this protein in events that precede prespliceosome formation. Data reported here as well as in the accompanying papers strongly implicate Sub2p in multiple steps of the spliceosome assembly process.