Definition of a spliceosome interaction domain in yeast Prp2 ATPase.
The Saccharomyces cerevisiae splicing factor Prp2 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. It contains three domains: a unique N-terminal domain, a helicase domain that is highly conserved in the DExD/H protein family, and a C-terminal domain that is conserved in spliceosomal DEAH proteins Prp2, Prp16, Prp22, and Prp43. We examined the role of each domain of Prp2 by deletion mutagenesis. Whereas deletions of either the helicase or C-terminal domain are lethal, deletions in the N-terminal domain have no detectable effect on Prp2 activity. Overexpression of the C-terminal domain of Prp2 exacerbates the temperature-sensitive phenotype of a prp2(Ts) strain, suggesting that the C-domain interferes with the activity of the Prp2(Ts) protein. A genetic approach was then taken to study interactions between Prp2 and the spliceosome. Previously, we isolated dominant negative mutants in the helicase domain of Prp2 that inhibit the activity of wild-type Prp2 when the mutant protein is overexpressed. We mutagenized one prp2 release mutant gene and screened for loss of dominant negative function. Several weak binding mutants were isolated and mapped to the C terminus of Prp2, further indicating the importance of the C terminus in spliceosome binding. This study is the first to indicate that amino acid substitutions outside the helicase domain can abolish spliceosome contact and splicing activity of a spliceosomal DEAH protein.
Edwalds-Gilbert
Gretchen
G
Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Kim
Dong-Ho
DH
Silverman
Edward
E
Lin
Ren-Jang
RJ
D000595:Amino Acid Sequence; D053487:DEAD-box RNA Helicases; D005690:Galactose / Q000378:metabolism; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012326:RNA Splicing / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism*
D029701:Saccharomyces cerevisiae Proteins; C066490:PRP2 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D005690:Galactose