Dominant negative mutants of the yeast splicing factor Prp2 map to a putative cleft region in the helicase domain of DExD/H-box proteins

Abstract

The Prp2 protein of Saccharomyces cerevisiae is an RNA-dependent ATPase required before the first transesterification reaction in pre-mRNA splicing. Prp2 binds to the spliceosome in the absence of ATP and is released following ATP hydrolysis. We determined what regions in Prp2 are essential for release from the spliceosome by analyzing dominant negative mutants in vivo and in vitro. We made mutations in conserved motif II (DExH) and motif VI (QRxGR) of the helicase (H) domain. Mutations that inactivated PRP2 had a dominant negative phenotype when overexpressed in vivo. To test whether mutations outside of the H domain could confer a dominant negative phenotype, we mutagenized a GAL1-PRP2 construct and screened for mutants unable to grow on galactose-containing media. Five dominant negative mutants were characterized; three mapped within the H domain and two mapped downstream of motif VI, indicating that an extended helicase domain is required for release of Prp2 from the spliceosome. Most mutants stalled in the spliceosome in vitro. However, not all mutants that were dominant negative in vivo were dominant negative in vitro, indicating that multiple mechanisms may cause a dominant negative phenotype. Structural modeling of the H domain of Prp2 suggests that mutants map to a cleft region found in helicases of known structure.