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Molecular and Cellular Biology, December 2004, p. 10101-10110, Vol. 24, No. 23
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.23.10101-10110.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Interaction between a G-Patch Protein and a Spliceosomal DEXD/H-Box ATPase That Is Critical for Splicing

Edward J. Silverman,1,2 Ayaka Maeda,2 Janet Wei,2 Paul Smith,3 Jean D. Beggs,3 and Ren-Jang Lin1,2*

Division of Molecular Biology,2 City of Hope Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, California,1 Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland, United Kingdom3

Received 22 June 2004/ Returned for modification 22 July 2004/ Accepted 10 September 2004

Prp2 is an RNA-dependent ATPase that activates the spliceosome before the first transesterification reaction of pre-mRNA splicing. Prp2 has extensive homology throughout the helicase domain characteristic of DEXD/H-box helicases and a conserved carboxyl-terminal domain also found in the spliceosomal helicases Prp16, Prp22, and Prp43. Despite the extensive homology shared by these helicases, each has a distinct, sequential role in splicing; thus, uncovering the determinants of specificity becomes crucial to the understanding of Prp2 and the other DEAH-splicing helicases. Mutations in an 11-mer near the C-terminal end of Prp2 eliminate its spliceosome binding and splicing activity. Here we show that a helicase-associated protein interacts with this domain and that this interaction contributes to the splicing process. First, a genome-wide yeast two-hybrid screen using Prp2 as bait identified Spp2, which contained a motif with glycine residues found in a number of RNA binding proteins. SPP2 was originally isolated as a genetic suppressor of a prp2 mutant. In a reciprocal screen, Spp2 specifically pulled out the C-terminal half of Prp2. Mutations in the Prp2 C-terminal 11-mer that disrupted function or spliceosome binding also disrupted Spp2 interaction. A screen of randomly mutagenized SPP2 clones identified an Spp2 protein with a mutation in the G patch that could restore interaction with Prp2 and enhanced splicing in a prp2 mutant strain. The study identifies a potential mechanism for Prp2 specificity mediated through a unique interaction with Spp2 and elucidates a role for a helicase-associated protein in the binding of a DEXD/H-box protein to the spliceosome.

* Corresponding author. Mailing address: 1450 E. Duarte Rd., Duarte, CA 91010. Phone: (626) 301-8286. Fax: (626) 301-8280. E-mail: rlin{at}coh.org.

Molecular and Cellular Biology, December 2004, p. 10101-10110, Vol. 24, No. 23
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.23.10101-10110.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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