ATP can be dispensable for prespliceosome formation in yeast

The first ATP-dependent step in pre-mRNA splicing involves the stable bindi ng of U2 snRNP to form the prespliceosome. We show that a prespliceosome-li ke complex forms in the absence of ATP in yeast extracts lacking the U2 sup pressor protein CUS2. These complexes display the same pre-mRNA and U snRNA requirements as authentic prespliceosomes and can be chased through the sp licing pathway, indicating that they are a functional intermediate in the s pliceosome assembly pathway. ATP-independent prespliceosome-like complexes are also observed in extracts containing a mutant U2 snRNA. Loss of CUS2 do es not bypass the role of PRP5, an RNA helicase family member required for ATP-dependent prespliceosome formation. Genetic interactions between CUS2 a nd a heat-sensitive prp5 allele parallel those observed between CUS2 and U2 , and suggest that CUS2 mediates functional interactions between U2 RNA and PRP5. We propose that CUS2 enforces ATP dependence during formation of the prespliceosome by brokering an interaction between PRP5 and the U2 snRNP t hat depends on correct U2 RNA structure.